Dear group, we are planning to embark on a project to attempt to quantify leukocyte surface antigens in blood samples from hospitalized patients. I would like to seek advice from the group given the technical and logistical difficulties that we face. Blood samples will be drawn from a hospital about two hours away from our research laboratory. I anticipate that some, but not all processing, can be done on site. Due to the long distance and the difficulty of the terrain, only one shuttle run per day can be done to bring the samples back to the main laboratory. Therefore, samples arriving to the main laboratory may be up to 24 hours old. Given this background, we are looking for field expediency without necessarily compromising too much on quality. I ask the following questions: 1. What is the optimal antibody staining light scatter combination to reliably identify granulocytes, monocytes and lymphocytes ? Some recommend CD14/CD45 while others seem to rely only on FSC and SSC. 2. What is the optimal method to anticoagulate and process the sample to quantitate for antigens such as integrins? I am not talking about what specific antibodies but such issues as choice of anticoagulant, presence or absence of calcium, fixation versus none, lysis or no lysis, etc. I am familiar with the work by McCarthy et al. (Cytometry 17:39-49,1994, J Immunol Methods 163:155-160,1993). These investigators recommend the use LDS 751 vs SSC to identify populations and argue for the use of PMSF as anticoagulant without fixation or lysis. You can reply directly to me or to the group as I think many others will be interested in the responses. Regards, -- Jose A. Stoute, MD Unit 64109, Box 401 USAMRU-Kenya APO AE 09831-4109 e-mail: stoutej@net2000ke.com Nairobi Tel 254-2-729303, Fax 254-2-714592 Kisumu Tel 254-35-22942, Fax 254-35-22903 Electronic Fax Service: 1-630-214-2008, 1-917-463-0373, 1-917-477-6048
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